Rathi Gangeswaran

Yes-associated protein (YAP) functions as a tumor suppressor in breast

Yes-associated protein (YAP) has been shown to positively regulate p53 family members and to be negatively regulated by the AKT proto-oncogene product in promoting apoptosis. On the basis of this function and its location at 11q22.2, a site of frequent loss of heterozygosity (LOH) in breast cancer, we investigated whether YAP is a tumor suppressor in breast. Examination of tumors by immunohistochemistry demonstrated significant loss of YAP protein. LOH analysis revealed that protein loss correlates with specific deletion of the YAP gene locus. Functionally, short hairpin RNA knockdown of YAP in breast cell lines suppressed anoikis, increased migration and invasiveness, inhibited the response to taxol and enhanced tumor growth in nude mice. This is the first report indicating YAP as a tumor suppressor, revealing its decreased expression in breast cancer as well as demonstrating the functional implications of YAP loss in several aspects of cancer signaling.

Molecular alterations in pancreatic carcinoma: expression profiling shows that dysregulated expression of S100 genes is highly prevalent

In order to expand our understanding of the molecular changes underlying the complex pathology of pancreatic malignancy, global gene expression profiling of pancreatic adenocarcinoma compared with normal pancreatic tissue was performed. Human cDNA arrays comprising 9932 elements were interrogated with fluorescence‐labelled normal and adenocarcinoma samples (nine tumours, three normal pancreata, and three cell lines). The data were analysed for differential gene expression, which was confirmed by serial analysis of gene expression (SAGE), digital differential display (DDD) analysis, and immunohistochemistry for selected cases. The array data were filtered to produce lists of a total of 75 genes significantly up‐regulated or down‐regulated in pancreatic adenocarcinoma. Two of those showing the highest differential were members of the S100 family of Ca‐binding proteins, namely S100P and S100A6, and therefore the S100 genes were studied in more detail. By immunohistochemical analysis of custom‐built, pancreas‐specific tissue arrays and commercially available, normal/cancer tissue arrays that included a wide variety of different tumour types, differential expression of S100P protein was found to be almost exclusive to pancreatic cancer. S100P could therefore represent a useful biomarker for pancreatic adenocarcinomas. Copyright

Expression of S100P and Its Novel Binding Partner S100PBPR in Early Pancreatic Cancer

S100P is a member of the S100 family of calcium-binding proteins and there have been several recent reports of its overexpression in pancreatic ductal adenocarcinoma (PDAC). We have used Far Western screening and in vitro interaction assays to identify and confirm a novel target protein for S100P. We have named this protein S100PBPR, and shown that its interaction with S100P is dependent on Ca(2+) or Mg(2+). S100PBPR was found to localize to cell nuclei where S100P is also present, and the two proteins co-immunoprecipitate. By in situ hybridization, S100PBPR transcript was found in islet cells but not duct cells of the healthy pancreas. Both S100P and S100PBPR were detected by quantitative real-time polymerase chain reaction in pancreatic intraepithelial neoplasia (PanIN) and PDAC samples, and in situ hybridization revealed the presence of S100PBPR transcript in malignant PDAC cells. These data suggest that an interaction between S100P and S100PBPR may be involved in early pancreatic cancer. S100P was further investigated in PanIN lesions and immunohistochemical analysis showed its expression to correlate significantly with increasing grade of PanINs, being found as early as PanIN-1 with more prevalent expression in PanIN-2 and -3. These data suggest that S100P can be added to the genetic progression model for PDAC.

Expression of the IAP protein family is dysregulated in pancreatic cancer cells and is important for resistance to chemotherapy

Pancreatic cancer is one of the most aggressive human tumors with a 5‐year survival rate of only 3% and a striking resistance to chemotherapy and radiotherapy. The search for new therapeutic approaches includes strategies exploiting the deregulation of apoptotic pathways commonly found in cancer cells. The IAP proteins are inhibitors of apoptosis that have altered activity in numerous cancer types and are implicated in resistance to chemotherapy, and therefore are potentially interesting as therapeutic targets. We investigated alterations in the expression of IAPs and their inhibitors in pancreatic adenocarcinoma by using real‐time PCR, in situ hybridization and immunohistochemistry. We found differential expression of various IAPs in this malignancy, and particularly we observed overexpression of cIAP‐2, survivin, livin and XIAP. We also looked for correlations between the expression of IAPs and resistance to paclitaxel, doxorubicin, CDDP and 5‐fluorouracil, and found that resistance to these drugs correlates most significantly with expression of cIAP‐2. Using RNAi to downregulate these proteins we further confirmed that the levels of cIAP‐2 and XIAP influence the response to the anti‐cancer drugs, although only marginally for 5‐FU. We conclude that anti‐tumor strategies based on the inhibition of particular IAPs can be useful in targeting pancreatic adenocarcinoma.

Unique protein kinase C profile in mouse oocytes: lack of calcium-dependent conventional isoforms suggested by rtPCR and Western blotting

rtPCR and Western blotting were used to determine which members of the PKC family are present in both immature and mature mouse oocytes. Using isoform‐specific PCR primers and antibodies PKC‐δ and ‐λ were detected while such techniques failed to observe the conventional isoforms of PKC‐α, ‐β, ‐γ. This isoform profile was confirmed using an alternative PCR strategy, which allowed discrimination of PCR products derived from conventional and novel PKC isoforms. In addition PKC‐ϵ, ‐η, ‐θ and ‐ζ were not detected by rtPCR. These results suggest that the predominant isoforms in oocytes are PKC‐δ and ‐λ.

Identification of genetic alterations in pancreatic cancer by the combined use of tissue microdissection and array-based comparative genomic hybridisation

Pancreatic ductal adenocarcinoma (PDAC) is characterised pathologically by a marked desmoplastic stromal reaction that significantly reduces the sensitivity and specificity of cytogenetic analysis. To identify genetic alterations that reflect the characteristics of the tumour in vivo, we screened a total of 23 microdissected PDAC tissue samples using array-based comparative genomic hybridisation (array CGH) with 1 Mb resolution. Highly stringent statistical analysis enabled us to define the regions of nonrandom genomic changes. We detected a total of 41 contiguous regions (>3.0 Mb) of copy number changes, such as a genetic gain at 7p22.2–p15.1 (26.0 Mb) and losses at 17p13.3–p11.2 (13.6 Mb), 18q21.2–q22.1 (12.0 Mb), 18q22.3–q23 (7.1 Mb) and 18q12.3–q21.2 (6.9 Mb). To validate our array CGH results, fluorescence in situ hybridisation was performed using four probes from those regions, showing that these genetic alterations were observed in 37–68% of a separate sample set of 19 PDAC cases. In particular, deletion of the SEC11L3 gene (18q21.32) was detected at a very high frequency (13 out of 19 cases; 68%) and in situ RNA hybridisation for this gene demonstrated a significant correlation between deletion and expression levels. It was further confirmed by reverse transcription–PCR that SEC11L3 mRNA was downregulated in 16 out of 16 PDAC tissues (100%). In conclusion, the combination of tissue microdissection and array CGH provided a valid data set that represents in vivo genetic changes in PDAC. Our results raise the possibility that the SEC11L3 gene may play a role as a tumour suppressor in this disease.

A Novel Therapeutic Regimen to Eradicate Established Solid Tumors with an Effective Induction of Tumor-Specific Immunity

Purpose: The efficacy of oncolytic viruses depends on multiple actions including direct tumor lysis, modulation of tumor perfusion, and stimulation of tumor-directed immune responses. In this study, we investigated whether a sequential combination of immunologically distinct viruses might enhance antitumor efficacy through the induction of tumor-specific immunity and circumvention or mitigation of antiviral immune responses. Experimental Design: The Syrian hamster as an immune-competent model that supports replication of both adenovirus and vaccinia virus was evaluated in vitro and in vivo. The antitumor efficacy of either virus alone or sequential combination of the two viruses was examined in pancreatic and kidney cancer models. The functional mechanism of the regimen developed here was investigated by histopathology, immunohistochemistry staining, CTL assay, and T-cell depletion. Results: The Syrian hamster is a suitable model for assessment of oncolytic adenovirus and vaccinia virus. Three low doses of adenovirus followed by three low doses of vaccinia virus resulted in a superior antitumor efficacy to the reverse combination, or six doses of either virus alone, against pancreatic and kidney tumors in Syrian hamsters. A total of 62.5% of animals bearing either tumor type treated with the sequential combination became tumor-free, accompanied by the induction of effective tumor-specific immunity. This enhanced efficacy was ablated by CD3+ T-cell depletion but was not associated with humoral immunity against the viruses. Conclusion: These findings show that sequential treatment of tumors with oncolytic adenovirus and vaccinia virus is a promising approach for cancer therapy and that T-cell responses play a critical role. Clin Cancer Res; 18(24); 6679–89. ©2012 AACR.

CEACAM6 attenuates adenovirus infection by antagonizing viral trafficking in cancer cells

The changes in cancer cell surface molecules and intracellular signaling pathways during tumorigenesis make delivery of adenovirus-based cancer therapies inefficient. Here we have identified carcinoembryonic antigen- related cell adhesion molecule 6 (CEACAM6) as a cellular protein that restricts the ability of adenoviral vectors to infect cancer cells. We have demonstrated that CEACAM6 can antagonize the Src signaling pathway, downregulate cancer cell cytoskeleton proteins, and block adenovirus trafficking to the nucleus of human pancreatic cancer cells. Similar to CEACAM6 overexpression, treatment with a Src-selective inhibitor significantly reduced adenovirus replication in these cancer cells and normal human epithelial cells. In a mouse xenograft tumor model, siRNA-mediated knockdown of CEACAM6 also significantly enhanced the antitumor effect of an oncolytic adenovirus. We propose that CEACAM6-associated signaling pathways could be potential targets for the development of biomarkers to predict the response of patients to adenovirus-based therapies, as well as for the development of more potent adenovirus-based therapeutics.

Lister Vaccine Strain of Vaccinia Virus Armed with the Endostatin–Angiostatin Fusion Gene: An Oncolytic Virus Superior to dl1520 (ONYX-015) for Human Head and Neck Cancer

Oncolytic viral therapy represents a promising strategy for the treatment of head and neck squamous cell carcinoma (HNSCC), with dl1520 (ONYX-015) the most widely used oncolytic adenovirus in clinical trials. This study aimed to determine the effectiveness of the Lister vaccine strain of vaccinia virus as well as a vaccinia virus armed with the endostatin-angiostatin fusion gene (VVhEA) as a novel therapy for HNSCC and to compare them with dl1520. The potency and replication of the Lister strain and VVhEA and the expression and function of the fusion protein were determined in human HNSCC cells in vitro and in vivo. Finally, the efficacy of VVhEA was compared with dl1520 in vivo in a human HNSCC model. The Lister vaccine strain of vaccinia virus was more effective than the adenovirus against all HNSCC cell lines tested in vitro. Although the potency of VVhEA was attenuated in vitro, the expression and function of the endostatin-angiostatin fusion protein was confirmed in HNSCC models both in vitro and in vivo. This novel vaccinia virus (VVhEA) demonstrated superior antitumor potency in vivo compared with both dl1520 and the control vaccinia virus. This study suggests that the Lister strain vaccinia virus armed with an endostatin-angiostatin fusion gene may be a potential therapeutic agent for HNSCC.

Modification of the Early Gene Enhancer-promoter Improves the Oncolytic Potency of Adenovirus 11

Oncolytic adenoviruses based on serotype 5 (Ad5) have several shortcomings, including the downregulation of its receptor in cancer cells, high prevalence of neutralizing antibodies and hepatotoxicity. Another adenoviral serotype, Ad11, could overcome these obstacles. Here, we show that human cancer cell lines express higher levels of the Ad11 receptor CD46, resulting in much better infectivity than Ad5. Surprisingly, only 36% (9/25) of the cell lines were more sensitive to Ad11- than to Ad5-mediated cytotoxicity. Investigations revealed that it was the transcription of Ad11 E1A, not CD46 expression or virus infectivity, which determined the cells sensitivity to Ad11 killing. Ad11 E1A mRNA levels have an effect on viral DNA replication, structural protein synthesis and infectious particle production. To test the hypothesis that increased E1A transcription would lead to improved Ad11 replication in Ad5-sensitive (but Ad11-less sensitive) cells, two Ad11 mutants (Ad11-Ad5-P and Ad11-Ad5-EP) were constructed where either the E1A promoter or enhancer-promoter, respectively, was replaced by that of Ad5. Ad11-Ad5-EP demonstrated increased E1A mRNA levels and replication, together with enhanced oncolytic potency in vitro and in vivo. This effect was found in both the Ad5-sensitive and Ad11-sensitive cancer cells, broadening the range of tumors that could be effectively killed by Ad11-Ad5-EP.